Functional Investigation of a Non-coding Variant Associated with Adolescent Idiopathic Scoliosis in Zebrafish: Elevated Expression of the Ladybird Homeobox Gene Causes Body Axis Deformation
Previously, we identified an adolescent idiopathic scoliosis susceptibility locus near human ladybird homeobox 1 (LBX1) and FLJ41350 by a genome-wide association study. Here, we characterized the associated non-coding variant and investigated the function of these genes. A chromosome conformation capture assay revealed that the genome region with the most significantly associated single nucleotide polymorphism (rs11190870) physically interacted with the promoter region of LBX1-FLJ41350. The promoter in the direction of LBX1, combined with a 590-bp region including rs11190870, had higher transcriptional activity with the risk allele than that with the non-risk allele in HEK 293T cells. The ubiquitous overexpression of human LBX1 or either of the zebrafish lbx genes (lbx1a, lbx1b, and lbx2), but not FLJ41350, in zebrafish embryos caused body curvature followed by death prior to vertebral column formation. Such body axis deformation was not observed in transcription activator-like effector nucleases mediated knockout zebrafish of lbx1b or lbx2. Mosaic expression of lbx1b driven by the GATA2 minimal promoter and the lbx1b enhancer in zebrafish significantly alleviated the embryonic lethal phenotype to allow observation of the later onset of the spinal curvature with or without vertebral malformation. Deformation of the embryonic body axis by lbx1b overexpression was associated with defects in convergent extension, which is a component of the main axis-elongation machinery in gastrulating embryos. In embryos overexpressing lbx1b, wnt5b, a ligand of the non-canonical Wnt/planar cell polarity (PCP) pathway, was significantly downregulated. Injection of mRNA for wnt5b or RhoA, a key downstream effector of Wnt/PCP signaling, rescued the defective convergent extension phenotype and attenuated the lbx1b-induced curvature of the body axis. Thus, our study presents a novel pathological feature of LBX1 and its zebrafish homologs in body axis deformation at various stages of embryonic and subsequent growth in zebrafish.
References
[1]
Weinstein SL. Natural history. Spine. 1999;24(24):2592–600. pmid:10635522. doi: 10.1097/00007632-199912150-00006
[2]
Pourquie O. Vertebrate segmentation: from cyclic gene networks to scoliosis. Cell. 2011;145(5):650–63. doi: 10.1016/j.cell.2011.05.011 pmid:21620133; PubMed Central PMCID: PMC3164975.
[3]
Hedequist D, Emans J. Congenital scoliosis: a review and update. Journal of pediatric orthopedics. 2007;27(1):106–16. doi: 10.1097/BPO.0b013e31802b4993 pmid:17195809.
[4]
Konieczny MR, Senyurt H, Krauspe R. Epidemiology of adolescent idiopathic scoliosis. Journal of children's orthopaedics. 2013;7(1):3–9. doi: 10.1007/s11832-012-0457-4 pmid:24432052; PubMed Central PMCID: PMC3566258.
[5]
Avivi E, Arzi H, Paz L, Caspi I, Chechik A. Skeletal manifestations of Marfan syndrome. The Israel Medical Association journal: IMAJ. 2008;10(3):186–8. pmid:18494229.
[6]
Shirley ED, Demaio M, Bodurtha J. Ehlers-danlos syndrome in orthopaedics: etiology, diagnosis, and treatment implications. Sports health. 2012;4(5):394–403. doi: 10.1177/1941738112452385 pmid:23016112; PubMed Central PMCID: PMC3435946.
[7]
Vialle R, Thevenin-Lemoine C, Mary P. Neuromuscular scoliosis. Orthopaedics & traumatology, surgery & research: OTSR. 2013;99(1 Suppl):S124–39. doi: 10.1016/j.otsr.2012.11.002 pmid:23337438.
[8]
Kouwenhoven JW, Castelein RM. The pathogenesis of adolescent idiopathic scoliosis: review of the literature. Spine. 2008;33(26):2898–908. doi: 10.1097/BRS.0b013e3181891751 pmid:19092622.
[9]
Brent AE, Tabin CJ. Developmental regulation of somite derivatives: muscle, cartilage and tendon. Current opinion in genetics & development. 2002;12(5):548–57. pmid:12200160. doi: 10.1016/s0959-437x(02)00339-8
[10]
Wallingford JB, Fraser SE, Harland RM. Convergent extension: the molecular control of polarized cell movement during embryonic development. Developmental cell. 2002;2(6):695–706. pmid:12062082. doi: 10.1016/s1534-5807(02)00197-1
[11]
Stemple DL. Structure and function of the notochord: an essential organ for chordate development. Development. 2005;132(11):2503–12. doi: 10.1242/dev.01812 pmid:15890825.
[12]
Ward K, Ogilvie J, Argyle V, Nelson L, Meade M, Braun J, et al. Polygenic inheritance of adolescent idiopathic scoliosis: a study of extended families in Utah. American Journal of Medical Genetics Part A. 2010;152A:1178–88. doi: 10.1002/ajmg.a.33145 pmid:20425822.
[13]
Ogura Y, Kou I, Miura S, Takahashi A, Xu L, Takeda K, et al. A Functional SNP in BNC2 Is Associated with Adolescent Idiopathic Scoliosis. American journal of human genetics. 2015;97(2):337–42. doi: 10.1016/j.ajhg.2015.06.012 pmid:26211971.
[14]
Sharma S, Londono D, Eckalbar WL, Gao X, Zhang D, Mauldin K, et al. A PAX1 enhancer locus is associated with susceptibility to idiopathic scoliosis in females. Nature Communications. 2015;6:6452. doi: 10.1038/ncomms7452 pmid:25784220.
[15]
Takahashi Y, Kou I, Takahashi A, Johnson Ta, Kono K, Kawakami N, et al. A genome-wide association study identifies common variants near LBX1 associated with adolescent idiopathic scoliosis. Nature Genetics. 2011;43:1237–40. doi: 10.1038/ng.974 pmid:22019779.
[16]
Kou I, Takahashi Y, Johnson TA, Takahashi A, Guo L, Dai J, et al. Genetic variants in GPR126 are associated with adolescent idiopathic scoliosis. Nat Genet. 2013;45(6):676–9. doi: 10.1038/ng.2639 pmid:23666238.
[17]
Fan Y-H, Song Y-Q, Chan D, Takahashi Y, Ikegawa S, Matsumoto M, et al. SNP rs11190870 near LBX1 is associated with adolescent idiopathic scoliosis in southern Chinese. Journal of Human Genetics. 2012;57:244–6. doi: 10.1038/jhg.2012.11 pmid:22301463.
[18]
Gao W, Peng Y, Liang G, Liang A, Ye W, Zhang L, et al. Association between common variants near LBX1 and adolescent idiopathic scoliosis replicated in the Chinese Han population. PloS One. 2013;8:e53234. doi: 10.1371/journal.pone.0053234 pmid:23308168.
[19]
Jiang H, Qiu X, Dai J, Yan H, Zhu Z, Qian B, et al. Association of rs11190870 near LBX1 with adolescent idiopathic scoliosis susceptibility in a Han Chinese population. European Spine Journal: official publication of the European Spine Society, the European Spinal Deformity Society, and the European Section of the Cervical Spine Research Society. 2013;22:282–6. doi: 10.1007/s00586-012-2532-4 pmid:23096252.
[20]
Londono D, Kou I, Johnson TA, Sharma S, Ogura Y, Tsunoda T, et al. A meta-analysis identifies adolescent idiopathic scoliosis association with LBX1 locus in multiple ethnic groups. Journal of medical genetics. 2014;51(6):401–6. doi: 10.1136/jmedgenet-2013-102067 pmid:24721834.
[21]
Jagla K, Dollé P, Mattei MG, Jagla T, Schuhbaur B, Dretzen G, et al. Mouse Lbx1 and human LBX1 define a novel mammalian homeobox gene family related to the Drosophila lady bird genes. Mechanisms of Development. 1995;53:345–56. pmid:8645601. doi: 10.1016/0925-4773(95)00450-5
[22]
Martin BL, Harland RM. A novel role for lbx1 in Xenopus hypaxial myogenesis. Development. 2006;133(2):195–208. doi: 10.1242/dev.02183 pmid:16339190.
[23]
Cheng L, Samad OA, Xu Y, Mizuguchi R, Luo P, Shirasawa S, et al. Lbx1 and Tlx3 are opposing switches in determining GABAergic versus glutamatergic transmitter phenotypes. Nature Neuroscience. 2005;8:1510–5. doi: 10.1038/nn1569 pmid:16234809.
[24]
Sch？fer K, Neuhaus P, Kruse J, Braun T. The homeobox gene Lbx1 specifies a subpopulation of cardiac neural crest necessary for normal heart development. Circulation Research. 2003;92:73–80. pmid:12522123. doi: 10.1161/01.res.0000050587.76563.a5
[25]
Watanabe S, Matsushita S, Hayasaka M, Hanaoka K. Generation of a conditional null allele of Lbx1. Genesis (New York, NY: 2000). 2011;49:803–10. doi: 10.1002/dvg.20739 pmid:21956886.
[26]
Schmitteckert S, Ziegler C, Kartes L, Rolletschek A. Transcription factor lbx1 expression in mouse embryonic stem cell-derived phenotypes. Stem cells international. 2011;2011:130970. doi: 10.4061/2011/130970 pmid:21941564; PubMed Central PMCID: PMC3175398.
[27]
Mennerich D, Braun T. Activation of myogenesis by the homeobox gene Lbx1 requires cell proliferation. The EMBO journal. 2001;20(24):7174–83. doi: 10.1093/emboj/20.24.7174 pmid:11742994; PubMed Central PMCID: PMC125799.
[28]
Brohmann H, Jagla K, Birchmeier C. The role of Lbx1 in migration of muscle precursor cells. Development (Cambridge, England). 2000;127:437–45. pmid:10603359.
[29]
Gross MK, Dottori M, Goulding M. Lbx1 specifies somatosensory association interneurons in the dorsal spinal cord. Neuron. 2002;34:535–49. pmid:12062038. doi: 10.1016/s0896-6273(02)00690-6
[30]
Gross MK, Moran-Rivard L, Velasquez T, Nakatsu MN, Jagla K, Goulding M. Lbx1 is required for muscle precursor migration along a lateral pathway into the limb. Development (Cambridge, England). 2000;127:413–24. pmid:10603357.
[31]
Ouellet J, Odent T. Animal models for scoliosis research: state of the art, current concepts and future perspective applications. Eur Spine J. 2013;22 Suppl 2:S81–95. doi: 10.1007/s00586-012-2396-7 pmid:23099524; PubMed Central PMCID: PMC3616476.
[32]
Castelein RM, van Dieen JH, Smit TH. The role of dorsal shear forces in the pathogenesis of adolescent idiopathic scoliosis—a hypothesis. Medical hypotheses. 2005;65(3):501–8. doi: 10.1016/j.mehy.2005.03.025 pmid:15913901.
[33]
Machida M, Dubousset J, Imamura Y, Iwaya T, Yamada T, Kimura J. An experimental study in chickens for the pathogenesis of idiopathic scoliosis. Spine. 1993;18(12):1609–15. pmid:8235839. doi: 10.1097/00007632-199309000-00007
[34]
Buchan JG, Gray RS, Gansner JM, Alvarado DM, Burgert L, Gitlin JD, et al. Kinesin family member 6 (kif6) is necessary for spine development in zebrafish. Developmental dynamics: an official publication of the American Association of Anatomists. 2014;243(12):1646–57. doi: 10.1002/dvdy.24208 pmid:25283277.
[35]
Fjelldal PG, Grotmol S, Kryvi H, Gjerdet NR, Taranger GL, Hansen T, et al. Pinealectomy induces malformation of the spine and reduces the mechanical strength of the vertebrae in Atlantic salmon, Salmo salar. Journal of pineal research. 2004;36(2):132–9. pmid:14962065. doi: 10.1046/j.1600-079x.2003.00109.x
[36]
Gorman KF, Tredwell SJ, Breden F. The mutant guppy syndrome curveback as a model for human heritable spinal curvature. Spine. 2007;32(7):735–41. doi: 10.1097/01.brs.0000259081.40354.e2 pmid:17414906.
[37]
Hayes M, Gao X, Yu LX, Paria N, Henkelman RM, Wise CA, et al. ptk7 mutant zebrafish models of congenital and idiopathic scoliosis implicate dysregulated Wnt signalling in disease. Nat Commun. 2014;5:4777. doi: 10.1038/ncomms5777 pmid:25182715; PubMed Central PMCID: PMC4155517.
[38]
Gray RS, Wilm TP, Smith J, Bagnat M, Dale RM, Topczewski J, et al. Loss of col8a1a function during zebrafish embryogenesis results in congenital vertebral malformations. Developmental Biology. 2014;386:72–85. doi: 10.1016/j.ydbio.2013.11.028 pmid:24333517.
[39]
Horike S, Cai S, Miyano M, Cheng JF, Kohwi-Shigematsu T. Loss of silent-chromatin looping and impaired imprinting of DLX5 in Rett syndrome. Nat Genet. 2005;37(1):31–40. doi: 10.1038/ng1491 pmid:15608638.
[40]
de Wit E, de Laat W. A decade of 3C technologies: insights into nuclear organization. Genes Dev. 2012;26(1):11–24. doi: 10.1101/gad.179804.111 pmid:22215806; PubMed Central PMCID: PMCPMC3258961.
[41]
Asakawa K, Suster ML, Mizusawa K, Nagayoshi S, Kotani T, Urasaki A, et al. Genetic dissection of neural circuits by Tol2 transposon-mediated Gal4 gene and enhancer trapping in zebrafish. Proc Natl Acad Sci U S A. 2008;105(4):1255–60. doi: 10.1073/pnas.0704963105 pmid:18202183; PubMed Central PMCID: PMCPMC2234125.
[42]
Kilian B, Mansukoski H, Barbosa FC, Ulrich F, Tada M, Heisenberg CP. The role of Ppt/Wnt5 in regulating cell shape and movement during zebrafish gastrulation. Mechanisms of Development. 2003;120:467–76. pmid:12676324. doi: 10.1016/s0925-4773(03)00004-2
[43]
Heisenberg CP, Tada M, Rauch GJ, Saúde L, Concha ML, Geisler R, et al. Silberblick/Wnt11 mediates convergent extension movements during zebrafish gastrulation. Nature. 2000;405:76–81. doi: 10.1038/35011068 pmid:10811221.
[44]
Lukowski CM, Drummond DL, Waskiewicz AJ. Pbx-dependent regulation of lbx gene expression in developing zebrafish embryos. Genome. 2011;54(12):973–85. doi: 10.1139/g11-061 pmid:22077099.
[45]
Sakuma T, Ochiai H, Kaneko T, Mashimo T, Tokumasu D, Sakane Y, et al. Repeating pattern of non-RVD variations in DNA-binding modules enhances TALEN activity. Scientific reports. 2013;3:3379. doi: 10.1038/srep03379 pmid:24287550; PubMed Central PMCID: PMC3843162.
[46]
Lours-Calet C, Alvares LE, El-Hanfy AS, Gandesha S, Walters EH, Sobreira DR, et al. Evolutionarily conserved morphogenetic movements at the vertebrate head-trunk interface coordinate the transport and assembly of hypopharyngeal structures. Dev Biol. 2014;390(2):231–46. doi: 10.1016/j.ydbio.2014.03.003 pmid:24662046; PubMed Central PMCID: PMC4010675.
[47]
Westfall TA, Brimeyer R, Twedt J, Gladon J, Olberding A, Furutani-Seiki M, et al. Wnt-5/pipetail functions in vertebrate axis formation as a negative regulator of Wnt/beta-catenin activity. The Journal of Cell Biology. 2003;162:889–98. doi: 10.1083/jcb.200303107 pmid:12952939.
[48]
Hayes M, Naito M, Daulat A, Angers S, Ciruna B. Ptk7 promotes non-canonical Wnt/PCP-mediated morphogenesis and inhibits Wnt/β-catenin-dependent cell fate decisions during vertebrate development. Development (Cambridge, England). 2013;140:1807–18. doi: 10.1242/dev.090183 pmid:23533179.
[49]
Gao B. Wnt regulation of planar cell polarity (PCP). Current topics in developmental biology. 2012;101:263–95. doi: 10.1016/B978-0-12-394592-1.00008-9 pmid:23140633.
[50]
Zhu S, Liu L, Korzh V, Gong Z, Low BC. RhoA acts downstream of Wnt5 and Wnt11 to regulate convergence and extension movements by involving effectors Rho kinase and Diaphanous: use of zebrafish as an in vivo model for GTPase signaling. Cellular Signalling. 2006;18:359–72. doi: 10.1016/j.cellsig.2005.05.019 pmid:16019189.
[51]
Purkiss SB, Driscoll B, Cole WG, Alman B. Idiopathic scoliosis in families of children with congenital scoliosis. Clinical orthopaedics and related research. 2002;(401):27–31. pmid:12151879. doi: 10.1097/00003086-200208000-00005
[52]
Maisenbacher MK, Han JS, O'Brien M L, Tracy MR, Erol B, Schaffer AA, et al. Molecular analysis of congenital scoliosis: a candidate gene approach. Human genetics. 2005;116(5):416–9. doi: 10.1007/s00439-005-1253-8 pmid:15717203.
[53]
Wright A, Charlesworth B, Rudan I, Carothers A, Campbell H. A polygenic basis for late-onset disease. Trends in genetics: TIG. 2003;19(2):97–106. pmid:12547519. doi: 10.1016/s0168-9525(02)00033-1
[54]
Andrews NC, Faller DV. A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic acids research. 1991;19(9):2499. pmid:2041787 doi: 10.1093/nar/19.9.2499
[55]
Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF. Stages of embryonic development of the zebrafish. Developmental dynamics: an official publication of the American Association of Anatomists. 1995;203:253–310. doi: 10.1002/aja.1002030302 pmid:8589427.
[56]
Kawakami K, Shima A. Identification of the Tol2 transposase of the medaka fish Oryzias latipes that catalyzes excision of a nonautonomous Tol2 element in zebrafish Danio rerio. Gene. 1999;240:239–44. pmid:10564832. doi: 10.1016/s0378-1119(99)00444-8
[57]
Kawakami K, Takeda H, Kawakami N, Kobayashi M, Matsuda N, Mishina M. A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish. Developmental cell. 2004;7:133–44. doi: 10.1016/j.devcel.2004.06.005 pmid:15239961.
[58]
Urasaki A, Morvan G, Kawakami K. Functional dissection of the Tol2 transposable element identified the minimal cis-sequence and a highly repetitive sequence in the subterminal region essential for transposition. Genetics. 2006;174:639–49. doi: 10.1534/genetics.106.060244 pmid:16959904.
[59]
Kwan KM, Fujimoto E, Grabher C, Mangum BD, Hardy ME, Campbell DS, et al. The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs. Developmental dynamics: an official publication of the American Association of Anatomists. 2007;236:3088–99. doi: 10.1002/dvdy.21343 pmid:17937395.
[60]
Bessa J, Tena JJ, de la Calle-Mustienes E, Fernández-Mi？án A, Naranjo S, Fernández A, et al. Zebrafish enhancer detection (ZED) vector: a new tool to facilitate transgenesis and the functional analysis of cis-regulatory regions in zebrafish. Developmental dynamics: an official publication of the American Association of Anatomists. 2009;238:2409–17. doi: 10.1002/dvdy.22051 pmid:19653328.
[61]
Sachdev SW, Dietz UH, Oshima Y, Lang MR, Knapik EW, Hiraki Y, et al. Sequence analysis of zebrafish chondromodulin-1 and expression profile in the notochord and chondrogenic regions during cartilage morphogenesis. Mechanisms of Development. 2001;105:157–62. pmid:11429291. doi: 10.1016/s0925-4773(01)00417-8
[62]
Alcaraz-Pérez F, Mulero V, Cayuela ML. Application of the dual-luciferase reporter assay to the analysis of promoter activity in Zebrafish embryos. BMC Biotechnology. 2008;8:81. doi: 10.1186/1472-6750-8-81 pmid:18954456.
